Evrogen introduces a novel cDNA depletion service allowing it to eliminate (deplete) selected sequences from cDNA library and thereby accelerate the discovery of the unknown genes
The recurring appearance of transcripts which have already been analysed during cDNA analysis often makes it an unacceptably time-consuming procedure.
The selective depletion of these from a cDNA library significantly accelerates the discovery of the unknown genes as well as functional screenings.
Evrogen introduces a novel cDNA depletion service allowing it to eliminate (deplete) selected sequences from cDNA library.
To do so Evrogen employs its thermostable duplex-specific nuclease (DSN) that cleaves ds-DNA specifically and not ss-DNA or RNA.
This specific feature, together with its capacity to function at relatively high temperatures, makes DSN ideal for the removal of ds DNA fraction from complex mixtures of nucleic acids and for the preparation of normalised and/or depleted cDNA libraries, the company says.
Evrogen cDNA depletion is adapted for use with smart technology prepared cDNA that is enriched with full length sequences and could be obtained both from poly(A)+ and total RNA, even if a small amount of starting material is available.
cDNA depletion is performed prior to library cloning and includes the following steps:.
Target cDNA is mixed with the excess of the synthetic driver DNA representing fragments of the genes to be eliminated, then it is denatured and allowed to re-anneal under hybridisation conditions.
During the hybridisation process, driver molecules form hybrids with the target sequences and remove them from the ss fraction.
On hybridisation completion, ds cDNA fraction is hydrolysed by DSN and ss DNA fraction is amplified using long-distance PCR.
Depletion procedure is accompanied by partial cDNA normalisation owing to abundant molecules also pass into ds form during hybridisation and is hydrolysed in the course of the DSN treatment.
Amplified cDNA is size fractionated and cloned into the vector of choice.
Evrogen says its service is an important element allowing researches to focus on the conceptual side of their scientific quest.
